Carotenoids include hydrocarbons (carotenes) and their oxygenated, alcoholic, derivatives (xanthophylls). Representative examples of carotenes include beta-carotene, alpha-carotene, and lycopene. Representative examples of xanthophylls include lutein, zeaxanthin, capsorubin, capsanthin, astaxanthin, and canthaxanthin.
Carotenoids are abundant in fruits and vegetables and have been studied extensively as antioxidants for the prevention of cancer and other human diseases. Among the dietary carotenoids, the focus has been on beta-carotene that has been established to play an important role in the prevention of various types of cancer.
More recent research has shown that other carotenoids, particularly the xanthophylls, posses strong antioxidant capabilities and may be useful in the prevention of diseases including cancer. For example, it has been reported that the consumption of lutein and zeaxanthin leads to a 40 percent reduction in age-related macular degeneration (Seddon et al., 1994, J. Amer. Med. Assoc. 272 (18): 1413-1420). It has also been reported that an increased level of serum carotenoids other than beta-carotene is associated with a lower incidence of heart disease (Morris et al., 1994, J. Amer. Med. Assoc. 272 (18): 1439-1441). The xanthophylls, because of their yellow to red coloration and natural occurrence in human foods, also find uses as food colorants. Thus there is an increasing need for substantially pure xanthophylls, which can be used as nutritional supplements and food additives.
Although present in many plant tissues, carotenoids free of other plant pigments are most readily obtained from flowers (marigold), fruits (berries) and root tissue (carrots and yellow potatoes). The hydrocarbon carotenes are typically present in uncombined, free from in chromoplast bodies within plant cells. Xanthophylls are typically present in plant chromoplasts as long chain fatty esters, typically diesters, of acids such as palmitic and myristic acids.
Although chemical processes for the synthesis of xanthophylls from commercially available starting materials are known, such processes are extremely time-consuming, involve multiple steps, and have not provided an economical process for the production of xanthophylls. A more economical route for the large-scale production of substantially pure xanthophylls is a process that extracts, isolates and purifies xanthophylls from natural sources. However, previous methods that isolate xanthophylls from plants use a number of organic solvents.
Previous investigators have also used a commercially available saponified marigold oleoresin, which contained free lutein, as a starting material, and then added the appropriate solvents to crystallize lutein from the saponified oleoresin (Tcyczkowski and Hamilton, Poultry Sci. 70 (3): 651-654, 1991; U.S. Pat. No. 5,382,714). The preparation of purified lutein difatty acid esters is also described in U.S. Pat. No. 4,048,203.
Methods for obtaining leaf xanthophylls are described in H. Strain, Leaf Xanthophylls, Carnegie institution, Washington, D.C. (1936). Among the techniques described, including those for obtaining xanthophylls occurring free in the leaves, Strain describes obtaining free xanthophylls from xanthophyll diesters present in the pods (calyx) of Physalis alkekengi.
In that latter preparation, at pages 99-104, the almost dried pods were cut into small sections with a meat grinder, and the pieces extracted with petroleum ether. The extract was concentrated to a small volume and the xanthophyll esters present were saponified with alcoholic potash. The alcohol was unnamed, but is presumed to be methanol from the preceding text. Water was added to the alcoholic solution to precipitate the xanthophylls. The precipitated xanthophylls were then crystallized several times from chloroform and a co-solvent such as methanol or petroleum ether, and then from pyridine.
The above procedure described by Strain has several disadvantages in producing a comestible xanthophyll. First, the hydrophobic petroleum ether used to extract the xanthophyll diesters is not removed prior to saponification and can be entrapped in the precipitated, hydrophobic xanthophylls. Second, use of a monohydric alcohol such as methanol or even ethanol can cause formation of water-insoluble fatty acid methyl or ethyl esters that can also be entrapped with the hydrophobic xanthophylls. Such entrapment may be the reason that so many recrystallization steps were required. Very cold temperatures such as -12.degree. C. and -70.degree. C. were also required for those recrystallizations.
The disadvantage of these methods is that the xanthophylls can retain some of the solvent(s) from which they are isolated and purified. In addition, these methods require the washing of the xanthophylls with more solvents. The solvents can be usually removed by drying the crystals at elevated temperatures. But in some instances, the solvent is difficult or impossible to remove. Traces of toxic organic solvents in the isolated, purified xanthophyll product make it unsuitable for human consumption. Another disadvantage of the use of a process that employs organic solvents is that such solvents are difficult to handle and present physical and chemical hazards. Still another disadvantage of that use is that organic solvents are a hazardous waste and present a disposal problem.
There is therefore a need for an economical means of production of an edible or comestible, substantially pure xanthophyll such as lutein or zeaxanthin in which the use of toxic or hazardous organic solvents is not employed. The process disclosed herein after provides such a comestible xanthophyll.